Gi/Go Couple Met–Enkephalin to Inhibition of Cholinergic and β-Adrenergic Stimulation of Lacrimal Secretion

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Abstract
To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent events and to identify the G proteins that couple met-enkephalin to inhibition of lacrimal secretion. Secretion of protein was measured in 3-day primary cultures of rabbit lacrimal acini exposed to vehicle, the cholinergic agonist carbachol (Cch), the beta-adrenergic agonist isoproterenol (Isop), vasoactive intestinal peptide (VIP), or forskolin (FSK) with or without the enkephalin analog D-ala2-met-enkephalinamide (DALA). In separate experiments, cells were pretreated with pertussis toxin or polyclonal antibodies against the alpha subunits of Gi/Go to determine the physiologic role of G proteins in met-enkephalin inhibition of the release of lacrimal protein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent protein kinase binding assay in lacrimal membranes in response to the same agonists used in the secretion studies. Cch resulted in a significant increase in protein release from cultured lacrimal acini. Increased secretion also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition of Cch-stimulated secretion was blocked by antibody specific to a common peptide sequence of Gialpha1 and Gialpha2 but was not blocked by anti-Gialpha1 antibody. The inhibitory effect on Cch-stimulated secretion was also blocked by anti-Gialpha3 and anti-Goalpha. Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation of secretion by immunoneutralization of Gialpha1/2 and Goalpha but not by immunoneutralization of Gialpha1 or Gialpha3. VIP, Isop, and FSK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any conditions. These results show that enkephalin inhibition of cholinergic and beta-adrenergic stimulation of secretion is mediated by Gi2, Gi3, and Go. The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of ion channels important for cholinergic and beta-adrenergic stimulation of lacrimal secretion.

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